Portable high gain fluorescence detection system

ABSTRACT

Disclosed is a compact, microprocessor-controlled instrument for fluorometric assays in liquid samples, the instrument having a floating stage with docking bay for receiving a microfluidic cartridge and a scanning detector head with on-board embedded microprocessor for controlling source LEDs, emission signal amplification and filtering in an isolated, low noise, high gain environment within the detector head. Multiple optical channels may be incorporated in the scanning head. In a preferred configuration, the assay is validated using dual channel optics for monitoring a first fluorophore associated with a target analyte and a second fluorophore associated with a control. Applications include molecular biological assays based on PCR amplification of target nucleic acids and fluorometric assays in general, many of which require temperature control during detection. Sensitivity and resistance to bubble interference during scanning are shown to be improved by use of a heating block with reflective mirror face in intimate contact with a thermo-optical window enclosing the liquid sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International PCT PatentApplication No. PCT/US2010/022581, which was filed on Jan. 29, 2010, nowpending, which claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/148,843, filed Jan. 30, 2009,which applications are incorporated herein by reference in theirentireties.

BACKGROUND

1. Field

The present invention relates to a compact fluorescence detectioninstrument with optics for use in assays performed in a microfluidiccartridge.

2. Description of the Related Art

Although the benefits of the use of fluorophores as probes for in-vitrodiagnostic assays are well known, the most commonly available forms ofequipment for such assays are large, complex to use, relatively slow andrely on expensive confocal optics. These attributes make much equipmentunsuitable for fully integrated “sample-to-answer” testing in remotelocales and on-site at the point of care, where such equipment isrequired to be rugged, fast, compact, inexpensive, and easy to use.Although automated nucleic acid amplification in a microfluidiccartridge was first proposed some years ago (see Wilding, U.S. Pat. Nos.5,304,487 and 5,635,358), detection of fluorescent assay targets outsidecontrolled laboratory conditions is still hampered by the lack ofportable and robust equipment. Two decades since their inception,molecular diagnostics are still relatively uncommon in the absence ofadvanced laboratory facilities because of these and other unsolvedproblems.

Needed to promote broader access to molecular diagnostics areself-contained assay systems designed to operate outside specializedlaboratory facilities. Nucleic acid assays are rapidly becoming the“gold standard” for the detection of many different disease types,including infectious diseases, because they offer both highersensitivity and specificity. Such assays have proven highly specific toa broad range of pathogenic conditions and are useful for trackinggenetic strains of a particular disease as is fundamental toepidemiology, for example in discriminating H5N1 avian influenza fromother types of influenza A or B, in determining whether a particularpathogen target is of a drug-resistant strain or not, and in detectingtoxigenic strains of an enteric isolate such as E. coli O157:H7.Fluorescence-based assays have also been shown to be useful formonitoring conditions such as diabetes, cardiopathies, coagulopathies,immunoassays in general, and for detection of endotoxin in foods or drugproducts for example. Improved equipment is particularly needed for thelarge numbers of remote health clinics in the developing world whereaccess to health care is limited and many infectious diseases areendemic, and health and life expectancy are poor.

In a typical fluorescence assay system, a fluorescent probe orfluorophore absorbs light having a wavelength or range of wavelengthsand becomes excited; and the fluorophore then emits a fluorescentsignal. The activity or inactivity of the fluorophore is indicative ofthe assay result. The emission signal has a wavelength or range ofwavelengths that is generally longer than the exciting light (but may beshorter as in “up-converting fluorophores”). A dichroic beam splitter orband-pass filter, or combination thereof, is then used to separate thefluorescent signal from other light, and the signal is passed to asensor. The sensor is often a photodiode, and generates an electricalsignal that can be used to score the assay. Qualitative and quantitativeassays using real time or endpoint fluorometry are feasible.

In such systems, a liquid sample is conveyed via a microfluidic channelinto a detection chamber or channel of a microfluidic cartridge where afluorescent probe admixed with or native to the sample is excited by anexcitation source. Controls may be run in parallel or multiplexed in theassay channel. Emitted light is measured to determine the presence orabsence of a target. A plurality of detection channels may be arrangedin the detection region of the microfluidic cartridge. Assays involvemaking one or more measurements of fluorescence; fluorophores may beused as markers for nucleic acid amplicons formed in an amplificationstep, or more generally for the presence or absence of a fluorescentassay target. Real time fluorometry, FRET, qPCR, thermal melt curves,kinetic and rate endpoints for assay scoring and validation are alsoknown in the art.

Prior art fluorescence detectors typically employ relatively expensiveoptical components (such as confocal optics, lasers and aspheric lenses)in order to pick up and localize fluorescent emissions present within amicrofluidic cartridge or microarray. WO 98/049543 to Juncosa, forexample, teaches three dichroic beam splitters in a single opticaltrain, one for controlling excitation source power and another forcontrolling reflectance signal; the third dichroic beam splitter is usedfor discriminating probe-specific fluorescent emission. One or morelenses serve to focus the excitation beam on the sample. Juncosa furtherteaches use of an aperture at the inlet of a photomultiplier and opticalobjective lens components of a confocal microscope for controlling animaging beam with a resolution of “microlocations” at about fiftymicrons. “By restricting the scope of the illumination to the area of agiven microlocation, or a fraction thereof, coupled with restricting thefield of view of the detector to the region of illumination, preferablythrough use of an aperture, significant improvements in signal-to-noiseratio may be achieved.” [p 7, lines 10-15]. These teachings are presagedby U.S. Pat. No. 3,013,467 to Minsky, U.S. Pat. Nos. 5,296,703 to Tsien,5,192,980 to Dixon, 5,631,734 to Stern, 5,730,850 to Kambara, and arereiterated in U.S. Pat. No. 6,614,030 to Maher and U.S. Pat. No.6,731,781 to Shams, among others. Maher uses lasers, fiber optics, aquartz plate and aspherical lenses with mini-confocal optical system inorder to optimize focusing and emission at a ten micron-sized spot atthe center of the microfluidic chamber [Col 3, lines 23-38; Col 7, lines7-16, 43-48 and 58-63].

Similarly, in U.S. Pat. No. 6,635,487, Lee affirms that focusing thecone of the excitation beam on the plane of the sample “provides thegreatest intensity to enhance analytical detection measurements on theassay chips.” [Col 1, lines 57-59]. This teaching thus encapsulates theprior art.

In a more recent filing, US Patent Application 2008/0297792 to Kimteaches that an image of an LED serving as a light source forfluorescence detection in a microfluidic chip is projected onto a sampleas an “optical spot” by an objective lens. The optical spot is focusedat the middle of the depth of a fluid in a chamber in the microfluidicchip [para 0018, 0067, claim 5]. Fluorescence emitted by the sample iscollimated as nearly as possible to parallel rays by the objective lensand focused on an avalanche photodiode. The requirement for highprecision in alignment relates to the dichroic mirror because thestopband will be shifted for light rays that do not enter the mirror ata 45° angle [para 0071], as is well known. Thus the teachings of Kimreflect the generally recognized state of the art.

In PCT Publication WO2008/101732 to Gruler, where is described afluorescence detector head for multiplexing multiple excitation anddetection wavelengths in a single light path, it is stated that, “Aconfocal measurement means that the focus of the illumination optics orthe source, respectively intrinsically is the same as the focus of thedetection optics or sensor, respectively.” [p7, lines 13-16]. Grulergoes on to state, “The confocal optics [of the invention] . . . secureshighest signal and lowest background intrinsic features of confocaldesign” [p32, lines 1-5], i.e., according to Gruler the highest possiblesignal and lowest noise are obtained with confocal optics.

While the consensus teaching of the prior art arose out of thespecialized use of confocal optics for epifluorescence microscopy, theteaching has been widely and uncritically applied to microfluidic,lateral flow, capillary electrophoresis and microarray applications.However, we have found that this approach is not well suited to liquidphase microfluidic diagnostic assays where detection of one or moremolecular probes in a fluid-filled channel is required. Due to effectssuch as photoquenching, thermo-convection, and the occasional presenceof bubbles or gradients in a fluid-filled channel, colocalizing thefocal point of the excitation beam and emission cone in the plane of thesample chamber can lead to unacceptable instability, loss of signal,quenching, noise, irreproducibility and overall loss of sensitivity inthe results. Because of the higher temperatures of PCR, for example,outgassing of reagents and sample is not an uncommon problem, andinterference from bubbles entrained in the liquid sample is a frequentproblem. The conventional approach also requires more expensive opticalcomponents and thus is disadvantageous for widespread applicationoutside advanced clinical laboratories.

A second problem is assay validation. Current standards for validationof infectious disease assays by PCR, for example, have come to rely onuse of spiked nucleic acid templates or more preferably, co-detection ofendogenous normal flora, for example ubiquitous non-pathogenicEscherichia coli in stools where pathogens such as Salmonella typhi orE. coli O157 are suspected. Another ubiquitous endogenous template ishuman 18S rRNA, which is associated with higher quality respiratory andblood samples. Co-amplification and detection of an endogenous templateensures confidence in the assay results but is difficult to achieve inpractice because of possible crosstalk between the fluorophores used asmarkers. When using high gain amplification, some level of crossover inthe spectra of the excitation and emission of fluorophores commonlyselected for multiplex PCR is typical and expected. Thus a solution thatwould isolate fluorescent signals with spectrally overlapping shouldersby using separate optical channels within a scanning detector headhaving shared low-noise electronics for downstream processing would be atechnological advance of benefit in the art.

A third problem is portability. Use of disposable cartridges has provedbeneficial because cross-contamination due to shared reagent reservoirsand shared fluid-contacting surfaces is avoided. However, configuring aprecision optical instrument platform for accepting disposablecartridges is problematic. Problems include inaccuracies and stackup inmechanical tolerances that affect cartridge alignment and detector headpositioning, the need for forming a highly conductive thermal interfacebetween the plastic disposable cartridges and heating sources in theinstrument, the need for sealing the pneumatic interface between controlservos on the apparatus and microvalves on the cartridge, and thenecessarily shorter light path available in a microfluidic cartridge(typically about or less than 1 millimeter), which without optimizationcan lead to loss in sensitivity. A simultaneous solution of theseinterlocking problems is only achieved by extensive experimentation anddevelopment, most often guided by trial and error in this highlyunpredictable art. Thus there is a need in the art for numerousimprovements, elements of which are the subject of the disclosureherein.

BRIEF SUMMARY

The present invention addresses the problem of reliable and sensitivedetection of fluorescent probes, tags, fluorophores and analytes in amicrofluidic cartridge in the presence of bubbles and other interferinginhomogeneities in a liquid sample, in a first aspect of the invention,by providing a reflective mirror face formed on a heating block thatcontactingly interfaces with a thermo-optical window on the underside ofthe detection channel or chamber containing the liquid sample. Themirror face is formed on the top surface of a heating block and contactsthe lower optical window of the detection chamber during use, avoidingthe complexity and expense of manufacturing an integral mirror on thebottom of each disposable cartridge, and allowing us to use thinner,more compliant films with lower resistance to heat transfer andtransparent optical characteristics for the thermo-optical window of thecartridge. The mirror face is optically flat and polished to improveboth heat transfer and fluorescence emission capture. A scanningobjective lens is positioned above an upper optical window on the top ofthe microfluidic cartridge. Excitation light is transmitted through boththe upper optical window and the lower thermo-optical window beforestriking the mirror and reflecting back. Direct and reflected emissionsare collected by the objective lens and focused on a detection sensorsuch as a photodiode, photocell, photovoltaic device, CMOS or CCD chip.

Also, and starkly in contrast to the teachings of the prior art, theproblem of fluorescence detection is shown to be solved by configuringthe optics so that the excitation optics are decoupled from the emissionoptics on a common optical path. By trial and error, when using the backmirror, we have found that it is advantageous to place the focal pointof the excitation cone near or behind the plane of the reflective mirrorand to independently position the emission cone so that emissions arepreferentially focused on the detection sensor. Surprisingly, decouplingincreases sensitivity, improves limits of detection, and reduces noiseor interference of bubbles and other inhomogeneities in the sample.

Contrary to the teachings of the prior art, we find that theconventional confocal localization of the excitation and emission signalis less effective in generating a robust signal over a wide range ofsample and operating conditions. Therefore, in one aspect of theinvention, it was found that optimization of signal detection may beimproved by displacement of the focal point of the excitation light fromthe plane of the sample to a point behind the sample, a technologicaladvance in the field of low cost optics for use with microfluidicfluorescence assays. Decoupling of excitation and emission optics fliesagainst decades of prior art dedicated to the principles (first espousedby Minsky in U.S. Pat. No. 3,013,467) that form the foundation ofconventional practice in confocal microscopy, epifluorescence detection,and microfluidic fluorescence assays. The prior art teachings have leadto the use of aspherical lenses, laser diodes, and precise parfocalalignment of the detection optics with the excitation optics. Incontrast, the optics required for delocalized focus of the excitationcone as described here are fortuitously of very low cost and do notrequire precision assembly or maintenance, as is desirable formanufacturing a low cost, portable instrument.

The mirror face on the top surface of the heating block under thedetection chamber is used to increase sensitivity by improving thelight-gathering capacity of the objective lens. As the objective lens isplaced closer to the detection chamber, a lens of defined angularaperture and numerical aperture becomes more efficient in collectingemissions. Without the back mirror, collection efficiency of a typicalsystem of the prior art is less than 2.5% (assuming for example a 5 mmplanoconvex lens). Adding a back mirror can improve this by as much as200%, and theoretically as much as 400%. And focusing the excitationbeam behind the sample chamber can add synergically to any gain insensitivity by increasing the excitation pathlength by using a mirror.We have found that this is especially advantageous in low aspect ratiomicrofluidic cartridges, where the optical path length on a z-axis of acartridge is typically sub-millimeter in length, a significant reductionrelative to a standard optical cuvette. Happily, this combination wasalso found to reduce interferences due to irregularities in the samplechamber such as the presence of small bubbles.

In one embodiment, the mirror is a chromed or polished metal surface onan aluminum or copper heating bock, and is also used to transmit heat orcooling for temperature controlled assays, thus achieving anothersynergy of design. In a preferred embodiment, the mirror is anelectropolished chrome surface on an optically flat aluminum block, thealuminum selected for its superior heat transfer characteristics andscaleable thermal inertia. The block is heated by a resistive heatingelement in contact with the base of the block. The smoothness andflatness of the mirror face favors optimal heat transfer. In this aspectof the invention, the mirrored face is the upper surface of the heatingelement used for example for FRET detection or thermal melting analysisof fluorescent probes for PCR amplicons. In one embodiment, a combinedapplication of the optical and thermal properties of the mirror-facedheating block is illustrated by construction of FRET melt curves takenby monitoring fluorescence while ramping the temperature of the assayfluid. In another embodiment, the mirror faced heating block is used toadjust or control a reaction temperature in the detection chamber of amicrofluidic card while the cartridge is scanned for fluorescenceemission. A mirror-faced heating block for use with microfluidiccartridges in real-time and temperature modulated fluorescence assaysdemonstrates a technical advance in the art.

According to another aspect of the present invention, we have employed ahigh gain multi-stage amplifier with noise elimination augmented throughthe use of downstream signal processing firmware compactly mounted inthe scanning head. Very high gain amplification and out-of-planedelocalization of the excitation light cone were found to be synergic inoptimizing assay discrimination and sensitivity, even in the presence ofbubbles which disrupt specular reflection from the mirror face behindthe liquid sample, and happily were implemented with no increase incost.

The complete optical path uses three lenses, the first for collimatingexcitation from a light source, the second for projecting the excitationsource onto the mirror and for collecting a fluorescent emission fromany fluorophore in the sample as collimated emissions, and the third forfocusing the emissions on a detector. Each fluorophore is opticallyisolated by a separate optical channel for measurement. A combination ofspectrally-specific LEDs, dichroic mirrors, and barrier filters are usedto achieve near monochromatic excitation light in each optical channel.The lenses and related optical components, including the dichroic mirrorintersecting the light path for separating excitation and emissionswavelengths and filters, are provided in a guiderail-mounted scanninghead that moves laterally across the detection chambers. To minimizenoise, the head also includes all electronic components for amplifyingthe signal and an on-board embedded microprocessor for analog anddigital signal processing. Even in the presence of bubble foaminterferences that defeat signal averaging and baseline subtractionmethods of data acquisition, assay scanning data from each opticalchannel may be accurately evaluated and reported by conversion to asingle bit output (ie. a 1 or a 0). This has been found to be a simpleand remarkably effective means for qualitative scoring for the presenceor absence of a signal from a particular fluorophore in a liquid samplemixture while the detector head is scanned over the detection chamber orchambers and across the mirror face.

The scanning head and rails are configured with a drive chain coupled toa stepper motor for accurate spatial resolution during scanning. Theentire microfluidic cartridge docking bay and optical bench is mountedin an instrument housing at a pitched angle, which we have foundadvantageous in decreasing bubble entrainment and improving ventingduring loading, wetout, and mixing operations on the microfluidiccartridge. In a preferred embodiment, the entire optical bench ismounted at an angle of about 15 degrees so that bubbles are displacedfrom the microfluidic circuitry, necessitating a complete suspensionmount for the floating optical bench and the docking bay, and aspring-biased clamping mechanism to ensure active formation of athermoconductive interface between on-board heating elements mounted inthe docking bay on the bottom of the optical bench assembly and theinsertable cartridge when the cartridge is loaded into the instrument. Asealed interface between the angled cartridge and a gasketed pneumaticinterface port must also be established during docking.

The detector head may be scanned across the detection chamber, orconversely, the microfluidic cartridge may be scanned across thedetector. As implemented here, the detector is mounted with a worm gear-or rack and pinion gear-driven stage under control of a stepper motor ontwin, paired guiderails. Samples may be scanned on demand bysynchronizing data acquisition with motor control; this may be performedusing an on-board or external host controller in communication with theembedded microprocessor in the detector head. Surprisingly, such adetector head with integral co-processor, mounted on a linear motionstage for sampling, filtering and averaging measurements on the fly,improved the capability of the system to validate and report assayresults despite many potential interferences.

It was expected that there will be variations in fluorescent intensityacross the microfluidic chamber, channel or detection field, which canbe several millimeters in width. These variations arise, for example, asa result of inhomogeneities in mixing, from differences in wellthickness, from imperfections in the optical windows, from smalltemperature variations across a heated detection well (which can causeaccompanying variations in hybridization-dependent fluorescence ofamplicons being detected, for example) and from bubbles or foam whicharise from degassing and mixing. It was found that these variations canbe minimized by signal digitization over a sampling transect across thedetection chamber using a threshold to discriminate positive andnegative test results.

In order to further provide noise elimination, extraneous noise isremoved by strobing the excitation beam at a frequency known to preventinterference by AC line fluctuations and other ambient electric or RFinterference; resulting in cleaner signal modulation, a modulated signalthat also can be filtered to remove the effects of any ambient lightleaking into the instrument housing. We have achieved this result byconfiguring the optics printed circuit board with a dedicatedco-processor, and using independent clock frequency and firmware, whichefficiently minimizes traffic on a databus connected to the hostinstrument. The on-board optical signal processor has dedicatedinstructions stored in EEPROM resident on the optics card, andsynchronizes pulses sent to the source LED with interrogation of thesensor photodiode at a frequency selected to limit electromagneticinterference. The firmware is designed to evaluate the difference insignal between strobe illumination bursts of excitation light andbackground between strobe bursts, and any background due to ambient orextraneous light is readily subtracted by this method.

The resulting optics modules are packaged in a sealed detector housingand can be expanded as a series of optics modules with multiple opticalchannels for simultaneously scanning multiple samples or fluorophores inseries or in parallel. Signal processing is routed to a singleco-processor embedded in the detector head, and from there to the hostinstrument controller. Thus the invention comprises single head/singlechannel embodiments, dual head/dual channel embodiments, andmulti-head/multichannel embodiments and may perform single and multiplexassays on single samples or on multiple samples in parallel.

The detectors were found to function well when housed as dual head andmulti-head detectors, where two or more channels in a single housingwere configured with fully independent optics, fluorophore-specificfilters, dichroic mirrors and source LEDs, reducing crosstalk betweenmultiple fluorophores. The head thus will optionally contain a pluralityof light sources mounted on a first circuit board and a plurality ofobjective lens and associated detectors mounted on a second circuitboard, and will collect signals for each of a plurality of fluorophoresindependently using shared signal processing capability and firmwareembedded in the detector head. To reduce noise, no analog signals aretransmitted from the detector head to the host instrument.

In this way, light sources for excitation in each channel can be matchedto the individual fluorophores. It is no longer necessary to providewhite light and an excitation filter to ensure a narrow pass beam ofexcitation light striking the sample. This simplification proved usefulin assay protocols calling for paired collection of “biplex” ormultiplex target and control signals. Where, as for FDA CLIA waiverrequirements, both target and control templates are amplified inparallel, a positive control signal must be present before an assayresult on a test sample can be reported or billed. In the absence of adetectable control signal, any target signal detected is not a validresult. In order to meet CLIA waiver requirements, it is necessary thatthe fluorescence detector be able to detect not only the presence, forexample, of a target infectious organism amplified by PCR but also of anendogenous human control organism co-existing with the target andamplified by the same PCR reaction or a PCR reaction conducted inparallel in the instrument, for example.

Such an approach requires that the fluorescent detector have thecapacity to determine the presence of both the target and the controlfluorophores as a “biplex” (“duplex”) amplification reaction mixture ina common detection chamber. A positive amplification control fluorophoreis typically used which has fluorescent excitation and emission spectrawhich is shifted (in wavelength) so as to be well resolved by selectiveband pass filters from the fluorescent excitation and emission spectrafor the target fluorophore (see FIG. 7 for example). However, accordingto the present invention, it proved possible to achieve superiorresolution by using a dual head detector and by scanning each detectionchamber twice in series, once with each optical channel, detecting firstthe control fluorescence signature, then the test sample fluorescencesignature—each scanning pass utilizing separate excitation and emissionoptics as described above.

A benefit was found by configuring these detectors with fully separatedand independent light paths. In this aspect of the invention, the use ofa duplex head design ensures that the presence of an amplificationcontrol fluorophore in a sample does not inadvertently create a signalin the target channel due to “crosstalk”. Such a situation would resultin the sample being classed as a “false positive”. Conversely, it isalso important that there is no crosstalk from the target channel to thecontrol channel, which is likely when multiple signals share a commonoptical path. Such a situation could result in the positiveamplification control being inadvertently deemed present, when this maynot be the case, and would lead to reporting of an invalid assay, anunacceptable outcome.

These principles are exemplified by the use of fluorescein or equivalentfluorophore as a molecular probe for the target, and Texas Red orequivalent fluorophores as a molecular probe for the control. A dualhead detector, with one detection channel optimized for detection offluorescein and the other detection channel optimized for Texas Red,each with separate excitation and detection optics, was found to besurprisingly sensitive, accurate and robust. The detector head was movedso as to position each optical channel in turn over the sample andseparate fluorescence readings were made. Surprisingly, this improvedresolution and minimized cross talk but did not contribute to highernoise or loss of sensitivity due to the mechanics of moving the head.Because excitation is not performed with white light, but is insteadperformed at a wavelength specific for an individual fluorophore,quenching of the second fluorophore is not an issue.

In another aspect of the invention, we have found that multiplemicrofluidic channels can be scanned by sequential traverse of amultihead detector across multiple sample wells, each head beingconfigured with independent optics for excitation and emission of asingle fluorophore. Although the excitation and detection optics areseparated for each optical channel, signal processing is performed incircuitry shared within the detector head.

Thus in yet another embodiment the invention provides a robustmulti-head independent channel fluorescent detection system for a pointof care molecular diagnostic assay which has a high degree ofspecificity directed towards the presence of both a target and annucleic acid amplification control co-existing in a common or paralleldetection chamber. The cleaner signals permit higher gain electronicamplification without a corresponding decrease in signal-to-noise ratio.Although a first embodiment in this invention describes a dual channeldetector (for the presence of a single target and a control), theinvention may also be applied to a fluorescent detection system havingmore than two channels, for example to detect a multiplex of targets anda control co-existing in a single detection chamber. Optionally, theheads may be positioned side-by-side, in an array, or radially, as in acylinder.

Use of the independent optical pathways fortuitously resulted in reducedneed for precision in assembly of lenses, dichroic beam splitters, andassociated filter elements, improving the manufacturability of theapparatus. Embodiments of the present invention incorporate inexpensivenon-precision optics, plastic lenses, a mirror on the heating blockbehind the sample window, moveable stage elements, strobed excitationand emission, noise suppression, on-board continuous signal processingover a movable detection field, and more than one optical channel forbiplex assay validation by use of paired target and controlfluorophores. Despite its high amplification gain, the instrument hasproven advantageously resistant to interferences such as electricalnoise and bubbles in the detection chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an instrument of the invention and amicrofluidic cartridge in the docking bay.

FIG. 2 is an animated view showing the insertion of the microfluidiccartridge in the docking bay.

FIG. 3A is a simplified representation of a floating stage with dockingbay, optical bench, and clamping mechanism for thermally contacting themicrofluidic cartridge with the heating module and scanning themicrofluidic cartridge.

FIG. 3B demonstrates conceptually that the floating stage, docking bay,optical bench and microfluidic cartridge are mounted in the instrumentchassis at a defined angle “theta” relative to the ground plane, wherethe ground plane is horizontal.

FIGS. 4A and 4B are front and rear interior perspective views fromabove, showing the suspension-mounted stage with docking bay, opticalbench, and scanning detector head.

FIG. 5A is a front interior perspective view from below the docking bay,showing the underside heating module and cooling fan.

FIG. 5B is a detail of the heating module with heating block elementsand mirror face.

FIGS. 6A and 6B are perspective views of the floating stage withinsertable cartridge in place in the docking bay. For clarity, thedocking saddle and accessory mounting elements have been removed.

FIG. 7 is a detailed view of the docking bay and floating stagesuspended from the underside of the docking saddle. The floating stageis fitted with a four point spring suspension.

FIGS. 8A and 8B are anterior subassembly views of the clampingmechanism. FIG. 8B illustrates the worm drive operation on the clampinggear rack.

FIGS. 9A and 9B are posterior subassembly views of the clampingmechanism. FIG. 9B illustrates the worm drive operation of the clampinggear rack and the platen arm.

FIG. 10 is a block diagram providing an overview of the functionalunits, software and firmware of the apparatus.

FIGS. 11A and 11B are perspective views of an insertable microfluidiccartridge for use with the apparatus of the invention.

FIG. 12 is an exploded view showing the internal components of amicrofluidic cartridge of FIG. 11.

FIG. 13 is a perspective view of a detector head with dual opticalchannels and electronically isolated circuit boards for excitation andemissions detection. One half of the housing is removed in order to viewthe internal components.

FIGS. 14A and 14B are schematic views of the internal optical componentsof a fluorescence detector with dual optical channels, heatingblock-mounted mirror and microfluidic cartridge. Excitation optics aremounted on one circuit board and detection optics on another to reducenoise interference.

FIG. 15 is a representation of an excitation cone and planoconvexobjective lens relative to the cartridge detection chamber andmirror-faced heating block.

FIG. 16 is a representation of emission collection with a planoconvexobjective lens at short working distance relative to the cartridgedetection chamber and mirror-faced heating block. Shown are primary andreflected fluorescent emissions.

FIG. 17 is a schematic representation of an optical pathway withdecoupled excitation and emission optics.

FIG. 18 is a block diagram of the detector head electronics used forcontrolling the fluorescent excitation, and receiving, processing, anddigitally communicating fluorescence emission signals to the hostinstrument.

FIGS. 19A and 19B are representations of raw input and digitized outputshowing digital removal of bubble interference.

FIGS. 20A and 20B show emission and excitation wavelengths for twofluorophores in a liquid sample, and illustrate dual head opticalisolation for removal of crosstalk.

FIG. 21A plots experimental results demonstrating enhancement of signaloutput by varying the height of the objective lens above the mirror.FIG. 21B graphs the integrated output signal strength with and withoutthe back mirror. Output signal was found to be optimized by focusing theexcitation beam at a focal point behind the mirror as shown in FIG. 15and collecting emissions at a shorter working distance as shown in FIG.16.

FIGS. 22 and 23A and 23B demonstrate a thermal melt curve of a molecularbeacon hybridized to an amplicon.

DETAILED DESCRIPTION

Although the following detailed description contains specific detailsfor the purposes of illustration, one of skill in the art willappreciate that many variations and alterations to the following detailsare within the scope of the claimed invention. The following definitionsare set forth as an aid in explaining the invention as claimed.

Definitions

“Angular aperture”—is the angle between the most divergent rays from asingle point that can enter the objective lens and participate in imageformation.

“Back focal length”—is defined for a lens with an incident beam ofcollimated light entering the lens as the distance L from the backsurface of the lens to the focal point of a cone of focused light. “Backfocal positions” indicates that non-collimated rays may be focused atalternate distances from the back of the lens by decoupling the optics.

Target analyte: or “analyte of interest”, or “target molecule”, mayinclude a nucleic acid, a protein, an antigen, an antibody, acarbohydrate, a cell component, a lipid, a receptor ligand, a smallmolecule such as a drug, and so forth. Target nucleic acids includegenes, portions of genes, regulatory sequences of genes, mRNAs, rRNAs,tRNAs, siRNAs, cDNA and may be single stranded, double stranded ortriple stranded. Some nucleic acid targets have polymorphisms, deletionsand alternate splice sequences. Multiple target domains may exist in asingle molecule, for example an immunogen may include multiple antigenicdeterminants. An antibody includes variable regions, constant regions,and the Fc region, which is of value in immobilizing antibodies. Targetanalytes are not generally provided with the cartridge as manufactured,but are contained in the liquid sample to be assayed; in contrast,“control analytes” are typically provided with the cartridge and areassayed in order to ensure proper performance of the assay. Spikedsamples containing target assay may be used in certain quality controltesting and for calibration, as is well known in the art.

Means for Amplifying: The grandfather technique was the polymerase chainreaction (referred to as PCR) which is described in detail in U.S. Pat.Nos. 4,683,195, 4,683,202 and 4,800,159, Ausubel et al. CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989), and in Innis et al., (“PCR Protocols”, Academic Press, Inc., SanDiego Calif., 1990). Polymerase chain reaction methodologies requirethermocycling and are well known in the art. Briefly, in PCR, two primersequences are prepared that are complementary to regions on oppositecomplementary strands of a target sequence. An excess of deoxynucleosidetriphosphates are added to a reaction mixture along with a DNApolymerase, e.g., Taq polymerase. If the target sequence is present in asample, the primers will bind to the target and the polymerase willcause the primers to be extended along the marker sequence by adding onnucleotides. By raising and lowering the temperature of the reactionmixture, the extended primers will dissociate from the template to formreaction products, excess primers will bind to the template and to thereaction products and the process is repeated. By adding fluorescentintercalating agents, PCR products can be detected in real time.

Other amplification protocols include LAMP (loop-mediated isothermalamplification of DNA) reverse transcription polymerase chain reaction(RT-PCR), ligase chain reaction (“LCR”), transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA), “Rolling Circle”, “RACE” and “one-sided PCR”,also termed “asymmetrical PCR” may also be used, having the advantagethat the strand complementary to a detectable probe is synthesized inexcess.

These various non-PCR amplification protocols have various advantages indiagnostic assays, but PCR remains the workhorse in the molecularbiology laboratory and in clinical diagnostics. Embodiments disclosedhere for microfluidic PCR should be considered representative andexemplary of a general class of microfluidic devices capable ofexecuting one or various amplification protocols.

Typically, nucleic acid amplification or extension involves mixing oneor more target nucleic acids which can have different sequences with a“master mix” containing the reaction components for performing theamplification reaction and subjecting this reaction mixture totemperature conditions that allow for the amplification of the targetnucleic acid. The reaction components in the master mix can include abuffer which regulates the pH of the reaction mixture, one or more ofthe natural nucleotides (corresponding to A, C, G, and T or U—oftenpresent in equal concentrations), that provide the energy andnucleotides necessary for the synthesis of nucleic acids, primers orprimer pairs that bind to the template in order to facilitate theinitiation of nucleic acid synthesis and a polymerase that adds thenucleotides to the complementary nucleic acid strand being synthesized.However, means for amplication also include the use of modified or“non-standard” or “non-natural” bases such as described in U.S. Pat. No.7,514,212 to Prudent and U.S. Pat. Nos. 7,517,651 and 7,541,147 toMarshall as an aid to detecting a nucleic acid target.

Means for detection: as used herein, refers to an apparatus fordisplaying an endpoint, ie. the result of an assay, and may include aninstrument equipped with a spectrophotometer, fluorometer, luminometer,photomultiplier tube, photodiode, nephlometer, photon counter,voltmeter, ammeter, pH meter, capacitative sensor, radio-frequencytransmitter, magnetoresistometer, or Hall-effect device. Magnifyinglenses in the cover plate, optical filters, colored fluids and labelledprobes may be used to improve detection and interpretation of assayresults. “Labels” or “tags” include, but not limited to, dyes such aschromophores and fluorophores; and chemoluminescence as is known in theprior art. QDots, such as CdSe coated with ZnS, decorated on magneticbeads, or amalgamations of QDots and paramagnetic Fe3O4 microparticles,are a convenient method of improving the sensitivity of an assay of thepresent invention. Fluorescence quenching detection endpoints are alsoanticipated. A variety of substrate and product chromophores associatedwith enzyme-linked immunoassays are also well known in the art andprovide a means for amplifying a detection signal so as to improve thesensitivity of the assay, for example “up-converting” fluorophores.Fluorescence and optical detectors may include photodiodes, photovoltaicdevices, phototransistors, avalanche photodiodes, photoresistors, CMOS,CCD, CIDs (charge injection devices), photomultipliers, and reversebiased LEDs. Detection systems are optionally qualitative, quantitativeor semi-quantitative.

“Molecular beacon”—is a single stranded hairpin-shaped oligonucleotideprobe designed to report the presence of specific nucleic acids in asolution. A molecular beacon consists of four components; a stem,hairpin loop, end-labelled fluorophore and opposite end-labelledquencher. When the hairpin-like beacon is not bound to a target, thefluorophore and quencher lie close together and fluorescence issuppressed. In the presence of a complementary target nucleotidesequence, the stem of the beacon opens to hybridize to the target. Thisseparates the fluorophore and quencher, allowing the fluorophore tofluoresce. Alternatively, molecular beacons also include fluorophoresthat emit in the proximity of an end-labelled donor.‘Wavelength-shifting Molecular Beacons’ incorporate an additionalharvester fluorophore enabling the fluorophore to emit more strongly.Current reviews of molecular beacons include Wang K et al, 2009,Molecular engineering of DNA:molecular beacons. Angew Chem Int Ed Engl,48(5):856-870; Cissell K A et al, 2009, Resonance energy transfermethods of RNA detection, Anal Bioanal Chem 393(1):125-35 and Li Y, etal, 2008, Molecular Beacons: an optimal multifunctional biologicalprobe, Biochem Biophys Res Comm 373(4):457-61. Recent advances includeCady N.C., 2009, Quantum dot molecular beacons for DNA detection.Methods Mol Biol 554:367-79.

Fluorescence nucleic acid assays include amplification with taggedprimers and probe-based detection chemistries. Fluorescent products canbe assayed at the end of the assay, or by measuring the amount ofamplified product in real time. While not limiting, TaqMan Probe(Applied Biosystems) which relies on displacement andpolymerase-mediated hydrolysis of a 5′ reporter dye with 3′ quencherconstruct, FRET hybridization probes, dual oligo FRET-based probes(Roche), minor groove binder-conjugated hybridization probes (MGBprobes, Applied Biosystems), Eclipse probes, Locked NA Probes(Exiqon/Roche), Amplifluor primer chemistries, Scorpions primerchemistries, LUX primers, Qzyme primers, RT-PCR, among others, are allsuitable in the present invention. Intercalation dyes may also be used.Reverse transcriptase is used to analyze RNA targets and requires aseparate step to form cDNA. Recent advances include Krasnoperov L N etal. 2010. Luminescent probes for ultrasensitive detection of nucleicacids. Bioconjug Chem 2010 Jan. 19 epub.

In addition to chemical dyes, probes include green fluorescent proteins,quantum dots, and nanodots, all of which are fluorescent. Molecules suchas nucleic acids and antibodies, and other molecules having affinity foran assay target, may be tagged with a fluorophore to form a probe usefulin fluorescent assays of the invention.

“FRET” (Fluorescence Resonance Energy Transfer)—is a fluorescencetechnique that enables investigation of molecular interactions. Itdepends on the transfer of energy from one fluorophore to anotherfluorophore (ie. a donor and a quencher) when the two molecules are inclose proximity such a when hybridized. Recent advances include CarmonaA K et al, 2009, The use of fluorescence resonance energy transfer(FRET) peptides for measurement of clinically important proteolyticenzymes, An Acad Bras Cienc 81(3):381-92.

Unless the context requires otherwise, throughout the specification andclaims which follow, the word “comprise” and variations thereof, suchas, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”. Referencethroughout this specification to “one embodiment”, “an embodiment”, “oneaspect”, or “an aspect” means that a particular feature, structure orcharacteristic described in connection with the embodiment or aspect maybe included one embodiment but not necessarily all embodiments of theinvention. Furthermore, the features, structures, or characteristics ofthe invention disclosed here may be combined in any suitable manner inone or more embodiments. “Conventional” is a term designating that whichis known in the prior art to which this invention relates. “About” and“generally” are broadening expressions of inexactitude, describing acondition of being “more or less”, “approximately”, or “almost” in thesense of “just about”, where variation would be insignificant, obvious,or of equivalent utility or function, and further indicating theexistence of obvious minor exceptions to a norm, rule or limit.

“Crosstalk”—in fluorescence imaging occurs when the excitation and/oremission spectra of two or more fluorophores (and/or autofluorescence)in a specimen overlap, making it difficult to isolate the activity ofone fluorophore alone.

Turning now to the figures, FIG. 1 is a perspective view of theinstrument 100 with a microfluidic cartridge 200 in the docking bay.Shown are membrane panel 104 and touch screen display surfaces 108 andcompact chassis or housing 106. Because all reagents are provided in themicrofluidic cartridge, the instrument has full standalone operability.FIG. 2 complements this exterior view by animating the insertion of themicrofluidic cartridge anterior nose 105 into the docking bay 103. Thedocking bay is suspension-mounted and tilted at an angle relative to theinstrument base, as will be discussed in more detail below.

The angled, tilted, floating stage with on-board optical bench anddocking bay is a distinctive feature of the instrument. This feature isintroduced conceptually in FIG. 3. A tilt sensor may be used inconjunction with the instrument host controller in order to ensure theproper angle is maintained for improved performance. The mounting angleof the inclined mounting plate determines the angle at which themicrofluidic cartridge is held during the assay. This angle “theta” hasbeen found to be advantageous in the range of 10-45 degrees from theground plane, more preferably 10-20 degrees, and is most preferentiallyabout 15 degrees. The angular mount has been found to relieve bubbleinterference that may be associated with deterioration in PCRamplification results, a technological advance in the art.

FIG. 3A is a schematic diagram of the primary optothermomechanicalsubsystems of the instrument. The floating stage 300 consists of atray-like chassis 301 that is suspended on an inclined plane by afour-point spring-mounted suspension (indicated by 302,303) and supportsa docking bay 103 for receiving a microfluidic cartridge 200 andscanning detector head 311 mounted on paired guiderails (308,309). Thecartridge is not part of the instrument 100, but interfaces with theinstrument after insertion into the floating docking bay 103.

During operation, the floating stage is clamped (indicated by 320)against inclined mounting plate (330) and engages contacting surfaces ofzone heating blocks (341,342,343,344) of a heating module 340 andassociated resistive heating elements and circuits. A fan 345 isprovided to dissipate excess heat during cooling. The inclined mountingplate is also provided with a pneumatic interface port 350 for sealedlydocking to the base of the microfluidic cartridge. Pneumatic pressure isdelivered to the cartridge through the pneumatic interface port from anintegral pneumatic distribution “manifold” or system embedded in theinclined mounting plate 330. The pneumatic manifold supplies negativeand positive pressure from sources mounted on the inclined mountingplate. A motherboard-mounted, programmable host controller directspneumatic driving pressure, vacuum, and control pulses to pumps andvalves on the cartridge via the internal manifold in the base plate 330and pneumatic interface port 350.

The detector head is motorized and scanning of the cartridge isperformed under the control of the central host controller. To scan thedetector head along paired guiderails (308,309) the host controllerengages a worm-gear driven by stepper motor 307. The detector head isfitted with an external window with objective lens 315 which scansoptical windows in the anterior nose 105 of the microfluidic cartridgeand collects raw optical signals. The detector head has its own embeddedmicroprocessor as described in FIGS. 10 and 18. The programmable hostcontroller also regulates temperature in one or more heating elements inthe heating module and a set of solenoid valves and positive pressureand vacuum pump reservoirs linked to the pneumatic interface. Theinstrument is supplied with a display panel and touch panel for userinteractions. Power input is flexible, and is optionally supplied by anAC adaptor, car adaptor, or from a rechargeable battery mounted underthe instrument. Also included are optional wireless IO and digital IOports.

FIG. 3B demonstrates conceptually that the floating stage 301, dockingbay 103, detector head 311, and microfluidic cartridge 200 are mountedin the instrument chassis 106 at a defined angle relative to the groundplane. Tilting the cartridge at an angle from the ground plane improvesventing during fluid loading and minimizes air entrainment duringwetting and mixing operations. Bubble accumulation, which interfereswith heat transfer and optical interrogation of assay results, isavoided by this and other innovations disclosed here. The inclinedmounting plate 330 establishes the angle of the floating stage 301,cartridge 200, and mechanical components of the clamping 800 and opticalscanning 310 subassemblies. We also found that bubble accumulationinterfered with nucleic acid amplification, and was limited by theangular mount of the stage.

As shown in FIG. 3B, the detector head is mounted in a clamshell housingwith mating half shells (312,313). The detector head slides on lateralguide rails 308 and 309 and is under host control of a stepper motor 307with worm drive. The floating stage chassis 301 is springedly mounted ina four-point suspension and has no direct connection to the inclinedmounting plate 330 until clamped. The clamping mechanism is indicatedhere figuratively by an arrow 320 and will be discussed in more detailbelow. All mechanical components of the clamping mechanism are attachedto the inclined mounting plate so that the entire docking bay andclamping mechanism are rotated at a fixed angle theta from the groundplane.

One of the two scanning guiderails 308 is readily visible in this view,and is supported at either end by the floating stage chassis or tray301. The docking bay (103) is indicated by a dotted line and marks theopening for insertion of the nose of the microfluidic cartridge underthe detector head 311, which scans from side to side as shown (doublearrow). The pneumatic interface port 350, shown here as a raisedplatform under the docking bay, obscures the position of the heatingmodule 340 and heating blocks 341-344 immediately inferior and in linewith the docking bay. Power conditioning, AC adaptors and batterystorage functions are mounted beneath the inclined mounting plate 330above the underside of the 360, which is designed to rest on a flatsurface.

FIGS. 4A and 4B are front and rear interior CAD views from above,showing the suspension-mounted stage with floating chassis 301 withoptical bench and detector head 311, and docking bay occupied by amicrofluidic cartridge 200. The floating stage is suspended from asaddle shaped support, docking saddle 400, which is rigidly bolted tothe inclined mounting plate 330. Also shown is a gear rack 401 thatprovides clamping pressure, as will be described in more detail below.In FIG. 4B, the guiderails (308,309) and stepper motor 307 of theoptical bench are readily recognized. The inclined mounting plate 330 ispopulated with pumps, vacuum and pressure storage tanks, solenoids andpneumatic control circuitry (not shown), but will not be described indetail here.

FIG. 5A is a front interior perspective view from below the docking bay,showing the underside heating module 340 and cooling fan 345. It can beseen that on insertion of the microfluidic cartridge 200 into thedocking bay, the heating module 340 is brought into alignment with theunderside of the cartridge.

FIG. 5B is a detail of the heating module 340 with heating blockelements (“thermal elements” 341,342, 343,344) and mirror face (500).The superior aspect of the heating module consists of one or moreheating blocks, each of which forms a thermal interface with a definedzone on the underside of the microfluidic cartridge for proper operationof the biochemical or molecular biological reactions that occur in theenclosed channels and chambers of the cartridge during the assay. Thesereactions can be as simple as immunobinding or hybridization, or ascomplex as nucleic acid amplification or enzymatic dehydrogenationcoupled to the formation or consumption of nicotinamide adeninedinucleotide and adenosine triphosphate, or cascading clotting factors,and generally require relatively stringent temperature control foroptimal reactivity and specificity. The heating blocks (341,342,343,344,although the invention is not limited to this configuration) may bespring-mounted and are urged upward in opposition to the downwardpressure of the clamping mechanism so as to establish high thermaldiffusivity contact zone for heat transfer. Each heating block is inthermal contact with a resistive (Coulombic) heating element, generallyby means of a compliant thermal pad for good thermal conductivity. Eachheating block contacts a thermal window in the microfluidic cartridge.Each window is generally a thin layer of a flexible plastic film, may beless than 3 mils in thickness, and most commonly of a complianttransparent material such as polyethylene terephthalate (Mylar®),although optionally of a cyclic polyolefin or polyimide with goodoptical transparency, while not limited thereto (see U.S. Pat. No.7,416,892, which is co-assigned), and also having good thermalconductivity. Thus in one embodiment the invention is a thermo-opticalinterface for reflective transillumination of a detection chamber in amicrofluidic card while controlling or modulating the temperature of aliquid sample in the detection chamber. This feature is of benefit forperforming a reaction or reactions associated with an analyte whilemonitoring an optical signal associated with the analyte. In oneexemplary application, thermal melt curves are used to verify FREThybridization results.

Also shown is the fan housing 345, which is used to dissipate heat fromheat sinks below the heating blocks and in PID control of temperature inthe blocks in combination with resistance heating circuits (not shown).

Heating block 341 in this case is modified by fabrication with apolished chromium mirror face 500 on the upper aspect which contacts andaligns with thermo-optical windows in the microfluidic card 200 duringthe assay. The thermo-optical window in this case corresponds to adetection chamber enclosed in the cartridge body. The mirror facereflects light from the detector head 311, which scanninglytransilluminates the cartridge, back into the objective lens 315, andalso reflects any fluorescent emission from the cartridge detectionchambers back into the detector head and from there to the detectionsensor, which is typically a photodiode, as will be discussed in moredetail below. Heating block 341 in this example differs from the otherheating blocks, and is generally machined from aluminum, then polishedand coated with an underlayer of copper under nickel before applicationof the chromium mirror face. Electropolishing and/or buffing may be usedto form a highly reflective optical finish on the chrome surface. Theoptically flat superior surface of the block aids in heat transfer andimproves sensitivity of fluorescence assays. Happily, the use of themirror permits simultaneous heating and optical interrogation of thefluid contents of the detection chamber, as is useful for example inoptically assaying melting curves.

Other heating zones may be modified similarly to permit opticalmonitoring with simultaneous temperature control or modulation. Theconfiguration of heating zones and mirrors may be modified or adaptedfor particular assay/cartridge requirements, and is not limited to theconfiguration shown here.

FIG. 5B also shows pneumatic interface port 350, here with ten outlets,each independently ported to a source of positive or negative pressurefrom the pneumatic distribution manifold of the host instrument andindependently under the control of a programmable host controller. Theseoutlets interface and seal to mated inlets in the underside of themicrofluidic card, and a timed pattern of intercommunicating pneumaticpressure, vacuum and pressure pulses are routed through the pneumaticinterface to drive and control the assay in the cartridge.

FIGS. 6A and 6B are perspective views of the floating stage subassembly300 with insertable cartridge in place in the docking bay 103. Forclarity, the docking saddle 400 and accessory mounting elements havebeen removed. It can be seen in FIG. 6A how the inferior surface of themicrofluidic cartridge 200 is shaped to be contacted with the matedsuperior surfaces of the heating module 340 of FIG. 5B. The cartridge issecured and supported under the floating stage 301 by two attachedlateral flanges 609 and 610, which are bolted in place and guideinsertion.

In FIG. 6B, four vertical posts (601,602,603,604) forming the maleelements of the four-point suspension 600 are apparent on the anteriorsection of the floating stage 300. These posts are fitted with coilsprings (603 a in FIG. 7) and inserted into cylindrical suspensionhousings (605, 606,607,608 in FIG. 7) formed with in the docking saddle400. They serve to suspend the floating stage 300 and optical scanningassembly as will be described in more detail in the next figure, FIG. 7.The entire optics bench and docking bay subassembly shown in FIGS. 6Aand 6B floats on this suspension and is rigidly brought into contactwith the rest of the instrument only on downward action of the clampingmechanism as will be shown in FIGS. 8 and 9.

FIG. 7 is an exploded view of the floating stage 300 with detector head311 docking bay 103 suspended from the underside of the docking saddle400. The floating stage is fitted with a four point spring suspensionwith coil springs (replicates of 603 a) on each of the four supportingposts (601,602,603,604). Each post is received in a mated suspensionhousing (605, 606,607,608) of the docking saddle. The microfluidiccartridge 200 is not shown, but it can be seen in this view that thedocking bay is configured for receiving the nose of the cartridge at theprojecting nose 515 of the docking bay, so that the cartridge rests onlower lateral flanges (609,610). Alignment pins (616,617) ensure thatthe docking bay seats true when pressed down against the heating module340. The posterior section of the floating stage, which contains theguiderails (308,309) and detector head 311 for fluorescence scanning, isfree of any support other then at the docking saddle and is cantileveredfrom the docking saddle during operation. The role of guiderails(308,309) in supporting motion of the scanning detector head 311 isapparent in this view.

The docking saddle is provided with brackets 712 and 713 for attachingthe clamping mechanism 800 as will be discussed below, and bar codereaders as are useful in automated operation. Linker arm 710 with slot711 is engaged by the clamping gear mechanism as discussed below and thefloating stage 300 raised or lowered as a single assembly. Dockingsaddle 400 and linker arm 710 also operatively fix the floating stage atthe theta angle of the inclined mounting plate.

FIG. 8A is a frontal view of the clamping mechanism. The bridging shapeof the docking saddle 400 is seen to rest above the anterior nose 515 ofthe floating docking bay 103. Immediately under the docking bay mouth isthe pneumatic interface port 350, visible between lateral flanges 609and 610 forming the channel for receiving the microfluidic cartridge200. During docking of the loaded cartridge, the function of theclamping mechanism is to urge the floating stage and spring-mountedchassis 301 with inserted cartridge downward onto the pneumaticinterface port and heating module as described above.

Docking saddle 400 is bolted on inclined mounting plate 330, andfloating stage chassis 301 is suspended on the four-point suspension600. The suspension springs apply a downward pressure on the floatingstage, which is opposed by the suspending action of clamping assembly800 in the raised position. When clamping the cartridge, clamp gearpiece 804 is driven by worm gear 801 and worm gear motor 802, drivingtravelling axle 803 in a downward arc. The axle pin 803 a is attached toa cam block (901, visible in FIG. 9B). Linker arm (710, visible in FIGS.7 and 9A) follows the cam-action of the clamping gear 401 and sliderblock 901 up or down on the four-point suspension. When disengaging thecartridge, the action is reversed. Worm gear motor 802 is run clockwise,raising pin 803 a and cam block 901, thus lifting the linker arm 710,which is part of the stage chassis assembly 301, and then reversed(double arrow). When the stage chassis is in the uppermost restingposition, the microfluidic cartridge can be removed from the instrument.Mechanical fiducials and alignment pins are used to register thecartridge in the instrument docking bay during the assay.

FIG. 8B is a detailed view of the front side of the clamp gear piece 804and worm drive gear 801, showing also cam follower surfaces 805 and 806on the anterior edge of the gear member that are used by pressureswitches to monitor the position of the gear and actuate coordinatedmechanical functions by the host controller. For simplicity, thepressure switches are not shown. Axle 810 is the center of rotation forthe piece and rotates on pin 810 a. Axle 803 travels during rotation ofthe gear piece, driving a slider block which engages with the stagechassis as shown in the following figure.

FIG. 9B is a detailed view of the rear side of the clamp gear piece 804and worm drive gear 801 showing the central axle 810 a and acentric camblock axle 803 a and cam slider block (901).

FIGS. 9A and 9B are mechanical drawings showing the action of theclamping mechanism assembly 800. The purpose of the clamp gear-drivencam is to raise and lower the floating stage 301. The clamping gearpiece 804 pivots on stationary axle 810, so that travelling axle 803scribes an arc upward or downward, propelling the linker arm 710 up ordown vertically (double arrow). Slider block 901 captive on pin 803 aslides left to right in a slot 711 in the linker arm (710, see FIG. 7)to accommodate the lateral vector of the motion of the clamp gear whileraising or lowering the floating stage 300. The upward movement of thefloating stage is opposed by springs 603 a as shown in the precedingfigures.

The mechanism illustrated here is not limiting, insofar as the inventioncan be realized in alternate ways, for example by clamping up from thebottom rather than down from the top, or by magnetically clamping ratherthan mechanically clamping. Other spring means may be selected from coilspring, leaf spring, torsion spring, helical spring, and alternativessuch as pneumatic canisters (e.g. gas springs) and elastomeric materialsor other equivalent means known in the art.

FIG. 10 is a block diagram providing an overview of the functionalunits, software and firmware of the apparatus. As described above andpresented schematically here, a floating stage (1000, dotted line)within the instrument supports a docking bay 1001 for receiving amicrofluidic cartridge and is provided with a scanning detector head1002. The scanning detector head contains subassemblies for providingexcitation light and sensors for detecting, amplifying and processingfluorescent emission signals under control of an embeddedmicroprocessor. Interfacing with the floating stage are a heating modulewith separately controllable heating zones under control of the hostcontroller, a pneumatics interface connected to pneumatic servos mountedon the base plate 330, which also serves as a pneumatic distributionmanifold, a wire harness connecting the stepper motor and the hostcontroller, and wiring harnesses for the clamp motor, and relatedsensors, including pressure switches for measuring the position of theclamp and the microfluidic cartridge, a barcode reader, and temperaturemonitors. Optionally, a tilt gauge is also supplied to measureinstrument orientation before controls are activated.

Power is supplied to all systems by a rechargeable battery, or by directconnection to an AC converter or to a DC source such as an automobile.

The host controller 1003 is mounted on a motherboard which also containsa touch pad panel for operation of the instrument and an LCD displaypanel. The instrument may transmit data to an outside network or devicevia a variety of digital serial I/O links, including a wirelessnetworking card. A special digital junction is provided for serviceaccess to the RAM registers and programming, which is software encodedin solid state ROM.

General instructions for operation of the instrument, such as thesequence of pneumatic pulses and valve logic required to operate aparticular microfluidic card having the capability to diagnose aparticular disease or pathology from a liquid sample, are provided byprogrammable software in the host instrument. If for example, thebarcode reader detects a particular microfluidic cartridge, the deviceis programmed to perform a particular assay and interpret and displaythe results in a designated format. However, the operation of theoptics, including modulation of source intensity, signal amplificationand filtering, is controlled by an embedded microprocessor on the sensorPCB within the detector head. Thus analog operations that are highlysensitive to noise are shielded in the detector head from the more noisyenvironment of the host instrument, and transmission of analog signalsfrom detector head to host A/D converters is completely avoided. Thisunconventional separation of functions has happily proved highlyadvantageous in reducing noise susceptibility of the instrument, as isneeded for full portability and field operation.

FIGS. 11A and 11B are perspective views of an insertable microfluidiccartridge for use with the apparatus of the invention. The cartridgeshown here consists of a housing 1102 and coverplate 1103 with internalworkings. Port 1104 is for receiving a liquid sample and anterior nose1105 is for inserting into the docking bay of the host instrument.Housing cutout 1101 is for exposing optical windows formed in internalsubcomponents of the cartridge, as shown in FIG. 12. Gasket 1106 is forsealedly interfacing with the pneumatic interface port 350 of the hostinstrument.

FIG. 12 is an exploded view showing the internal components of amicrofluidic cartridge of FIG. 11. While not limiting in scope by thissingle embodiment, this particular cartridge 1100 is designed for PCRwith FRET or molecular beacon detection. An optical window 1101 on thenose of the housing 1102 inserts into the host instrument and aligns theoptical windows of the FRET or molecular beacon detection chambers 1201of the microfluidic inboard circuit card 1200 with the optical pathscanned by the detector head. Added microfluidic processing related tosample preparation is supplied on outboard card 1204. All liquidreagents are enclosed in sealed frangible pouches 1206 and are dispensedwhen needed under pneumatic control. Other reagents are provided on-cardin dry form. Fluid waste is sequestered in an adsorbent batting 1207that is sealed in place under the plastic coverplate 1103. The detailsare beyond the present scope, but microfluidic circuit 1200 withinternal microfluidic channels and wells for thermocycling andamplifying a nucleic acid target includes detection chambers 1201 forFRET detection of any resultant amplicon. The cartridge as shown is adisposable cartridge. Gasket material 1106 serves as a single-usesealing gasket between pneumatic control ports on the microfluidiccartridge and a corresponding pneumatic control manifold and interface350 on the inclined mounting plate 330.

FIG. 13 is a perspective view of a dual channel detector head 1300 withtwo optical channels and electronically isolated circuit boards(1301,1302) for excitation and for emissions detection respectively. Inthis view, the upper half of the housing 1303 is removed in order toshow the internal components of the detector head. The dual channels aremarked by objective lenses (1310, 1330) and optic pathways A and B (openarrows). The SMD LED excitation light sources (1311, 1331) are mountedon a source LED printed circuit board (1301), which is connected atright angles to sensor PCB (1302) via an edge-type resistivepin-connector (1304). The photodetection components are mounted on thesensor PCB (1302). A Faraday cage element (1306) is used to shield thephotodiodes (1317) and (1337) and surrounding high gain amplificationcircuitry.

Fluorescent excitation is provided in the target channel (Arrow A) by asurface mounted LED (1331) which is chosen to match the excitationspectrum of the target fluorophore. Source LED (1331) emits a divergentlight beam, and the radiated light beam is then collimated by sourceexcitation lens (1332). Source lens (1332) is a planoconvex lens havingits flat surface facing the LED. The collimated light beam may then bepassed through an excitation bandpass filter (1333), the purpose ofwhich is further explained in the description associated with FIG. 20.The collimated, filtered excitation light beam is then reflected from adichroic mirror element or beamsplitter (1334), which is installed at aforty-five degree angle to the incident beam, and is passed through aplanoconvex objective lens (1330) and through an external window in thedetector housing (Arrow A). After passing lens 1330, the excitationlight is focused through a detection chamber (not shown, see FIGS.14-17) embedded in a microfluidic cartridge, which contains a sampleliquid with any target fluorophore. The path length of the excitationlight through the sample liquid is doubled by use of a back mirrorbehind the microfluidic cartridge. The target fluorophore is excited bythe incident light beam. The emission of the fluorophore is generally ata longer wavelength than the excitation wavelength and is shifted by anamount equal to the Stokes shift of the target fluorophore.

A portion of the returning emission from the target fluorophore in thedetection chamber is collected by planoconvex sampling lens 1330 and iscollimated before striking dichroic mirror 1334. Optionally, a Fresnellens may be use to further reduce the working distance between the lensand the sample so as to optimize collection of emitted light, which isfurther enhanced by back mirror mounted on a heating block behind thedetection chamber. Because dichroic beamsplitter 1334 has a wavelengthcutoff between the excitation and emission wavelength, the dichroicmirror 1334 now acts as a pass-band beam splitter for the emittedfluorescent light beam and a stopband filter for the excitation light.It transmits the emitted fluorescent light while reflecting reflectedexcitation light and any ambient light entering the light path throughthe objective lens window. Emitted light passing through the dichroicbeamsplitter 1334 then passes through an emission filter 1335, thepurpose of which is further explained in the description associated withFIG. 20. Light exiting emission filter 1335 then passes throughplanoconvex sensor lens 1336, where it is focused onto the surface of aphoto-sensor 1337 which is surface mounted to PCB 1302 and is protectedfrom electrical noise by Faraday cage 1306.

The above described optical pathways are repeated in a second (control)channel having control excitation LED 1311, planoconvex excitation lens1312, excitation filter 1313, dichroic beamsplitter 1314, objective lens1310, control emission filter 1315, planoconvex sensor lens 1316, andcontrol photodiode 1317. Outputs from both photodiodes are amplified bythree-stage trans-impedance amplifiers built into the board next to thephotodiodes and grounded to an embedded microprocessor on the sensor PCBvia carefully shielded pins from the amplifiers.

In one embodiment, as exemplified by the use of fluorescein and TexasRed as fluorophores, excitation LED 1331 is a 470 nm LED with band-passexcitation filter 1333 for delivering essentially monochromatic light of485±12 nm used for the target channel and a 590 nm LED 1311 with bandpass filter 1313 was used for the control channel. The excitation LEDsare modulated or “strobed” on and off using a strobe rate of 130 Hz soas to filter AC power-related noise at 50 or 60 Hz and at harmonicfrequencies associated with fluorescent overhead illumination, alsofiltering phantom signal related to stray ambient light and electricalnoise that may be present at 30 or 60 Hz. Local feedback sensors areused to monitor and stabilize source LED output intensity. Detectionmonitoring of fluorophore emission is coordinated with movement on railsof the detector head under power of a stepper motor controlled by a hostcontroller. An embedded microprocessor and associated circuitry in thedetector head is provided with RAM memory, ROM memory, an A-D converter,a three-stage trans-impedance amplifier, and signal processing andcommand sequence firmware to handle these functions.

Each of the photo-sensors 1317 and 1337 are mounted on a common PCB1302. The output signal legs from each of these photo-sensors areconnected directly to the first stage of respective tri-stagetrans-impedance amplifiers (not shown). PCB 1302 makes extensive use ofhardware noise-reduction components, in particular an embedded groundplane and a Faraday Cage 1306 to minimize the unwanted effects of any RFor electromagnetic interference on the input signals. The combination ofthe use of these hardware noise-reduction elements with a digital signalprocessing (DSP) method, leads to a detector design which is essentiallyimmune from the effects of unwanted noise.

FIGS. 14A and 14B are schematic views of the internal optical componentsof a fluorescence detector head 1300, showing the external opticalinterface with optical windows in a microfluidic cartridge 1402 and backmirror 1400 mounted behind the cartridge on the surface of a heatingblock 1410 that is used to control or ramp the temperature in detectionchambers enclosed in the cartridge. Unconventionally, multipleindependent optic pathways or “channels” are formed in a singledetection head and share electronic PCBs and downstream signalprocessing circuitry, but excitation optics are mounted on one circuitboard and detection optics on another to reduce noise interference. Thetwo boards are electrically coupled by a corner mounted pin junction1304 and are electronically isolated.

Shown in FIG. 14A is the optical transition for the excitation of afluorophore in a detection well (1403 a or 1403 b) embedded within amicrofluidic cartridge 1402. The head is a scanning head and movesacross microfluidic cartridge 1402 (double arrow). Light from excitationLED 1331 on PCB 1301 is collimated by lens 1332 and made essentiallymonochromatic by band-pass filter 1333. Any fluorophore or fluorophoresin detection well 1403 a (whether the control or the target fluorophore)are excited by incident light 1420 focused on the sample by objectivelens 1330. In FIG. 14B, the emission of the fluorophore(s) is collectedby objective lens 1330 and transmitted to sensor 1337 after passingthrough dichroic beamsplitter 1334, emission filter 1335 and sensor lens1336. Sensor 1337 is in direct electrical contact with the base of ahigh gain transistor that amplifies the output signal and is shielded ina Faraday cage 1306. The emitted fluorescent light is generally at alonger wavelength according to the Stokes shift of the fluorophore,enabling the emitted light to pass through dichroic bandpass mirror 1334and emission band-pass filter 1335 without losses. Mirror face 1400 isused to increase the amount of excitation light on the target, doublingthe excitation path length, and to improve emission collectionefficiency. The light returned from sample chamber 1403 a to objectivelens 1330 is thus a mixture of emitted and reflected fluorescence 1421and reflected excitation light 1420. Light traps (not shown) areprovided to capture stray reflections. Reflected light 1420 does notpass dichroic mirror 1334 and is returned to the source, and does notinterfere with the measurement of emission intensity at sensor 1337. Theoptic elements of a single channel, including excitation source, sourcecollimating lens, excitation filter, dichroic mirror, objective lens,excitation filter, sensor lens, and detector with amplifier make up anoptics module having an essentially monochromatic source wavelength anda highly specific sensor for detecting fluorescence at a particularwavelength characteristic of the target (or control) fluorophore. Oneoptics module or channel may be used for an assay target, the othermodule for a control channel. Tandem mounted optics channels may be usedto collect data on a plurality of fluorophores, where electricalprocessing is shared by common embedded microprocessor beforetransmission to the host instrument. Optionally additional channels maybe use. Each channel shares the two PCB but has separate optics.

The microfluidic cartridge 1402 is movable (double arrow) relative todetector head 1300 and motorization of the detector head or cartridgetray or mounting chassis permits scanning: a transect across cartridge1402 permits measurements to be made on sample chambers 1403 a and 1403b, for example. By using multiple detection optics modules mountedside-by-side in a detector head, the sample chambers can be scanned formultiple fluorophores in series.

According to one embodiment, the excitation electronics are mounted on aprinted circuit board (1301) and the detection electronics are mountedon a second PCB (1302). An edge-connector 1304 electrically joins theboards. Faraday cage 1306 protects the sensor and associated high gainamplifier from stray electromagnetic noise. Mirror 1400 is fabricated onthe upper surface of heating block 1410, which also functions in heattransfer and controls the temperature of the sample fluid during theassay. The temperature of heating block 1410 can be ramped, for exampleas in a FRET melt determination under control of the host controller.

FIG. 15 is a representation of a planoconvex objective lens 1500 andexcitation cone 1501 relative to the cartridge detection chamber 1403and heating block 1410 with mirror face 1400. The excitation cone isformed by diverging rays illuminating the lens so that the distance L2′is greater than the native focal length L2 of the lens. By convention,the native back focal length L2 of the lens is determined usingcollimated light. Shifting the focal position is termed “decoupling”.

Interposed between the lens 1500 and the mirror face 1400 is amicrofluidic cartridge 200 with detection chamber 1403. The detectionchamber is bounded by an upper optical window 1502 and a lowerthermo-optical window 1503.

In operation, the intervening volume is taken up by a liquid sample,shown here with two entrained bubbles 1505. The focal cone is seen toreflect from the mirror face, forming a real image (1510, solid rays) ofthe source in the detection chamber and a virtual image (1511, dottedrays) of the source below the mirror face. The back focal position L2′is thus generally equal to or greater than the distance between the lensand the mirror. Excitation light striking the mirror is reflected as afocused beam in the fluid volume of the detection chamber, thus doublingthe length of the light path of the excitation light through the sampleand increasing the excitation fluorescence yield. The back focalposition L2′ is not equal to the back focal length L2 of lens 1500; thetwo are decoupled, generally by illuminating the lens with a divergentbeam from the source.

FIG. 16 is a representation of the objective lens of FIG. 15 in emissioncollection mode. Shown are primary and reflected fluorescent emissions(solid and dotted lines from a real image 1510 and a virtual image1511). Again shown are bubbles in the chamber 1403. Rays striking theplanar back surface of the lens will be collimated and transmitted to adetector. The quantity of fluorescence signal captured depends on theangular and numerical apertures of the objective lens 1500. The nativeback focal length of the lens 1500 is L2. The back focal length and backfocal position of the lens (L2 versus L2′) can be manipulated or“decoupled” by repositioning the source as shown below.

FIG. 17 is a schematic representation of an optical pathway 1700 withdecoupled excitation and emission optics. In this figure, excitationrays are shown as solid lines and emission rays are shown as dottedlines. Aspects of integration of the emission and detection elementsinto an integrated optical system are discussed. Here the light source1701, may be an LED as shown, an SMD LED without lens housing andreflector ring, an SLED, a pumping laser diode, a ridge-waveguide (FabryPerot) laser diode, a tuneable laser, and so forth, such source ofillumination preferably having a narrow bandwidth and serving as adirected source of collimated light. LEDs of various narrow bandwidthsare available, for example with peak emission at 630 nm (red), 470 nm(blue), 525 nm (green), 601 nm (orange), 588 nm (yellow) and so forth.While LEDs may also be used if desired, but generally the LED output ismatched with the fluorophore of the target in the assay and an optionalexcitation filter (not shown) may be used to sharpen the bandpass asrequired. Light output of light source 1701 is transmitted by sourcelens 1702, shown here as a planoconvex lens, although molded aspheresmay also be used, and optionally passed through a excitation filter (notshown) before striking dichroic mirror element 1704, where theexcitation beam is redirected to objective lens 1705. The excitationcone striking sample fluid volume 1720 in microfluidic cartridge 1402 isshown with solid lines. Unconventionally, the focal point of theexcitation cone has been projected past the sample chamber and backmirror 1400 by moving the source 1701 closer to the source lens 1702(ie. shortening distance L1′ in order to increase distance L2′, wheredistance L1 would be the native back focal length of lens 1702). Asdistance L1′ is shortened, the source rays striking the objective lensare caused to diverge, thus increasing distance L2′. The native backfocal length of objective lens 1705 is L2 and emissions from afluorophore in the sample chamber and reflected rays from the mirror arecollected as a virtual image of the fluorophore that enter the objectivelens in a cone having a focal point decoupled from the focal point ofthe excitation light (which is focused behind the mirror). The reflectedvirtual image of a population of fluorophores in the chamber is boundedby dotted lines crossing the heating block 1410. Fluorescent emissionswithin the focal length L2 are effectively collimated by the objectivelens; are transmitted through dichroic mirror 1704, bandpass filter1706, and are then focused by sensor lens 1707 onto sensor 1708, whichis generally mounted on a PCB or other solid support 1709. Lens 1707 hasa back focal length L3 that generally is equal to back focal length L2of objective lens 1705. However, a larger lens 1707 may be used tobetter utilize the surface area of the sensor 1707, which is for examplea photodiode or CCD chip. Optimization of signal may require independentadjustment of each lens according to these principles.

The excitation light emerging from objective lens 1705 and the emissionlight entering objective lens 1705 are operably decoupled in differentfocal cones (L2′ versus L2 respectively). A distance separates theactual focal plane of the excitation light and the native focal lengthof the objective lens, which can capture light in a broad plane oforigin of the fluorescent emissions when excited using a mirror and anextended focal position of the excitation cone 1501. This phenomenon,termed “decoupling” was found to increase capture of fluorescentemissions when a back mirror is used, and controverts earlier teachingsin favor of the confocal approach of the prior art.

While the teachings of the prior art strongly support making theexcitation and emission confocal, there is in fact an hithertofor unseenadvantage in decoupling the focus of the source from the emission coneand using a back mirror 1400. Emitted light arises from a greater areaand depth throughout the sample cartridge, thus overcoming any lack ofsignal from dead spots or inhomogeneities as would be due to smallbubbles, unmixed areas, or quenched probe. Greater reliability isachieved at the expense of some selectivity in the excitation at thepoint of focus. This is a technological advance in the art.

To summarize, generally, L2′ may be greater than L2 and L3. L1advantageously may be configured so that the cone of excitation lightfalls behind the sample chamber 1403 and most preferentially close to orbehind the back mirror 1400. In a preferred embodiment, the focal pointof the excitation cone falls on or behind the back mirror. The objectivelens is configured, generally, so that emitted light is efficientlycollected and collimated for projection onto the detection sensor by asymmetrical cone of emitted light from sensor lens 1707 (i.e. L2=L3).

Accordingly, in another embodiment the apparatus of the inventionemploys lenses configured so that excitation optics and the emissionoptics are decoupled. In a first embodiment of this apparatus, the lightsource is positioned at a distance L1′ from the source lens, whereL1′<L1, whereby the excitation optics and emission optics are decoupledby transitioning the excitation cone to a focal position L2′ at orbehind the mirror face, such that L2′>L2. In a second embodiment, thesource lens is configured to form a diverging beam of light incident onthe objective lens, thereby positioning the excitation cone at a focalposition L2′, whereby L2′>L2.

Advantageously, L2 is configured to be symmetrical to L3 (i.e., L2=L3),so that the operation of detector 1708 is optimized and robust. Thesensor photodiode is preferentially configured to be large enough withreference to the cone of focus of lenses 1705 and 1707 to accommodatesome degree of misalignment without loss of assay validity.

Performance is improved not only in assays where differentiation of apositive or negative assay result is required, but also in assays weresome level of quantitation is required, as for example schizont ormerozoite copy number in the case of Plasmodium falciparum. It should berecalled that the original purpose of confocal optics, as articulated byits inventor, Minsky in 1957 (U.S. Pat. No. 3,013,467), was to create athree-dimension image of a thick solid specimen by rastoring a focalpoint of excitation across and through the specimen (xyz axis rastoring)while monitoring emission only from the area of the specimen where theexcitation cone is focused at any given time. In contrast, in a fluidmixing specimen that is generally homogeneous, an opposite effect isdesired, that of measuring the cumulative fluorescence of the entirespecimen, and suppressing any localized variance in that fluorescence.Thus by reformulating the problem, we have been able to design a noveloptical system with back mirror, with decoupling of the focal plane ofexcitation and emission, with the happy result that fluorescencedetection is more sensitive and robust in the presence of occasionaloptical interferences.

FIG. 18 is a block diagram of the detector head electronics and opticsused for controlling the fluorescent excitation and for receiving,processing, and digitally communicating fluorescence emission signals tothe host instrument. Optical channels are again identified by openarrows A and B. Channel A is taken as a control channel and channel B asa target analyte channel, but the roles are interchangeable. Theelectronic functional blocks in each channel, source excitation circuits(1811,1831) and sensor circuits (1817,1837) are identical and are drivenby detection optics control circuitry of the embedded microprocessor1841, which has a dedicated on-board instruction set as firmware,typically as a socketed EEPROM chip 1842. The circuit board 1801 for thesource LEDs can support multiple LEDs, and the circuit board 1802 forthe sensor circuit can support multiple photodiodes. Each photodiode isintimately associated with a multiple stage high gain amplifier(1818,1838) and the two circuit boards are electronically isolated withseparate grounds.

The stepper motor and worm drive module controls scanning of thedetector head as directed by the host controller. Thus the detector headoperates as a self-contained optics and signal analysis module whilescanning under control of the host controller. The host also handles thedata tabulation and display functional block (1820), includingpreparation of test result reports and any I/O functions.

Within the detector head, each of the LEDs associated with sourceexcitation circuits (1811, 1831) are modulated by a square wave at afrequency of 130 Hz. The reason for this modulation is related to noisereduction measures from the following potential noise sources:

-   -   1. 50/60 Hz mains    -   2. 100/120 Hz second mains harmonic from the fluorescent lights.    -   3. Third and higher harmonics of 50/60 Hz.    -   4. Differential frequencies (rumble) of 130 Hz and all the above        of the    -   5. Photodiode sensor, first stage feedback resistor and        amplifier electrical noise. This noise is wide band white noise.

In order to retrieve the useful signal from the noisy source, thefollowing methods are employed:

-   -   1. Fast sampling and averaging of taken data in order to avoid        aliasing and at the same time limit noise bandwidth.    -   2. Modulation of the LED light at 130 Hz and correlation with        the detected fluorescence signal at 130 Hz in order to reject        all uncorrelated components. The 130 Hz modulating frequency was        selected to provide at least 10 Hz difference from the 50/60 Hz        mains and its harmonics (mainly 100, 120, 150 and 180 Hz).    -   3. Further filtering and processing of the correlated data to        eliminate electromagnetic noise from mains power supplies of        either 50 Hz or 60 Hz or harmonics thereof.

The advantage of using the embedded microprocessor 1841 with thefluorescence detector is that a proprietary method of digital signalprocessing (DSP) may be programmed into the firmware of an embeddedmicroprocessor in the detector to eliminate noise before transferring adigitized signal to the host controller.

The excitation LEDs are modulated at 130 Hz with 100% AM modulationdepth (on/off). The modulating frequency was selected to providesufficient separation from 50/60 Hz and its harmonics (50, 60, 100, 120,150, 180 Hz, et seq.). Any intermodulation product frequency is at least10 Hz and can be filtered out during signal processing. The LEDs aredriven by FAN5612 LED-drivers. Each driver is capable of sinking currentup to 120 mA (40 mA on each of three outputs) at the required frequency.

The host controller 1800 is responsible for the operator interface,including display of results, and for operation of mechanical andpneumatic functions required to perform an on-cartridge assay anddetector head scan. The embedded microprocessor 1841 in the detectorhead is responsible for controlling the excitation and detectioncircuits, which are electronically isolated on separate PCB boards(1801,1802) and for signal filtering, and has its own instruction setwhich is programmable as a socketed EEPROM chip 1842 on the sensorboard. The clock frequency of the embedded detector head microprocessoris used to strobe the excitation LEDs and to synchronize pulsecollection in the sensor diodes. A separate clock in the host controlleris used to drive the stepper motor during scanning. Multiple excitationand detection optics can be housed in a single detector head so thatsignal excitation and fluorescent emission detection can be multiplexedin the embedded processor. We have found that on-board packaging ofsignal processing achieves a low noise environment with improvedsignal-to-noise ratio and sensitivity by minimizing signal pathlengthsand permitting effective use of faraday shielding where necessary, suchas around the sensor diode leads and at the junction between theexcitation and sensor circuit boards.

Isolation of dual channels proved an advantageous means to implementmultiplex assays where target fluorophore and internal controlfluorophore are mixed in a common liquid sample. By separating targetand control channel optics, crosstalk that could lead to false positivetests or test rejection due to invalid control results was eliminated.

The host controller also controls pneumatic valve logic and pulse trainsfor operating diaphragm pumps in the microfluidic cartridge during theassay, any resistive or Peltier-type heating elements associated withthermal cycling of the sample, and optionally may perform melt curves inthe detection chamber. Other optional components include fiducials foraligning detector heads and a bar code reader for sensing informationprinted on the insertable microfluidic cartridges.

The host controller program with program coding means is designed toperform all the steps of a fluorescence assay process and to transformand format a signal or other data from the fluorescence detector into amachine-readable or graphical report of significance to the user. Theprogram can be integrated into the fluorescence detector instrument asshown here or can be connected to it through data lines or wirelessinterfaces as part of a network, intranet or internet. Generally, aserial asynchronous communications interface is provided forcommunication with the host controller on the instrument motherboard oron an external network.

Similarly, results, data, error codes, status updates, and so forth canbe sent via common electronic interfaces and data lines such as USB,RS232 or FireWire and via a wireless transmission system such asIR-transmission, Bluetooth, GSM, GPRS, RSID, etc. Programming,reprogramming, calibration, and recalibration as well as systemdiagnosis of the device is possible via common electronic interfaces anddata lines such as USB, RS232 or FireWire and via a wirelesstransmission system such as IR-transmission, Bluetooth, GSM, GPRS, RSID,etc.

The apparatus can be configured for wavelengths in the UV, visibleregion, and near infrared spectrum. For applications in fluorescencemode, which is one of the preferred operating modes, the device can beconfigured for specific fluorescence dyes with excitation spectrum inthe UV and visible spectrum and for emission in the UV, visible and nearinfrared spectrum. While a red shift is more typical, up-convertingfluorophores may also be used. Today available light sources filters andavailable dyes allow for customizing in the range of 300 to 900 nm.

FIGS. 19A and 19B are representations of raw input and digitized outputshowing digital removal of bubble interference. In FIG. 19A, output(1900) from the photodiode after amplification is represented. The levelof noise is generally low, but signal deteriorates at 1901 and 1902 dueto the presence of two small bubbles in the detection chamber (see FIG.15), for illustration. The processor applies a threshold to the outputsignal and scores the signal “high” (i.e. a one) if the signal is abovethe threshold and “low” (i.e. a zero) if the signal is below thethreshold. Since no signal output can be above the threshold except inthe presence of a fluorophore matched to the emission optics andfilters, any positive signal (1903) is a positive assay for the presenceof fluorophore. The threshold comparator can be adjusted based onexperience with clinical samples in the assay. Thus a “1-bit” digitaltransformation of the scanning image data removes any interference frombubbles. We have found surprisingly that when a fluorophore is presentbut multiple bubbles fill the chamber, light refracted around or throughthe bubbles will result in a positive signal. The system is thus veryerror resistant and robust for qualitative testing, such as is needed indiagnostic assays for infectious disease. The signal comparator is adigital function of the microprocessor and firmware embedded in thedetector head and is independent of host controller function.

Should the presence of a positive fluorescence signal indicate anegative assay result, the system can be easily configured to score thetest that way. Thus the use of a 1-bit digital transformation is aremarkably simple and effective solution for the presence of bubbles ina microfluidic assay. Because of the nature of the mixing and heatingoperations in microfluidic assays, and the frequent use of surfactants,the presence of bubbles is not uncommon. The systems described here usea combination of physical methods (venting, tilting of the stage, wetoutunder low dead volume conditions, accentric channels between mixingchambers) and signal processing methods to achieve robust assayperformance such as is needed for reliable operation outside thecontrolled environment of a clinical laboratory.

FIGS. 20A and 20B show the excitation and emission spectra of a typicalsystem of mixed fluorophores for the target and control, hereillustrated by fluorescein and Texas Red. Shown in FIG. 20A, curve 2001is the excitation spectrum for fluorescein (dotted line); curve 2002 isthe emission spectrum (solid). Shown in FIG. 20B, curve 2003 is theexcitation spectrum for Texas Red (dotted line); curve 2004 is thecorresponding emission spectrum (solid). Here control is Channel B (FIG.20B) and target is Channel A (FIG. 20A), but the assignment isarbitrary.

Boxed area ExA indicates the wavelength band that is allowed to passthrough the target excitation bandpass filter 1333 (cf FIG. 13). Box EmAindicates the wavelength band that is allowed to pass through the targetemission bandpass filter 1335. Box ExB indicates the wavelength bandthat is allowed to pass through the control excitation bandpass filter1313. Box EmB indicates the wavelength band that is allowed to passthrough the control emission bandpass filter 1315. The boxes indicatethe presence of stopbands on either side of the maxima. It can be seenfrom FIGS. 20A and 20B that, given the spectra for these twofluorophores and optical filters having the passband characteristicsconfigured as shown, the following error conditions are corrected:

-   -   a. Long wavelength excitation light from the target LED 1331        (greater than the wavelength of the LED peak excitation) cannot        be mistakenly confused for target fluorescent emission, due to        these longer wavelengths being cut off by the LED excitation        filter 1333.    -   b. Long wavelength excitation light from the control LED 1311        (greater than the wavelength of the LED peak excitation) cannot        be mistakenly confused for control fluorescent emission, due to        these longer wavelengths being cut off by the LED excitation        filter 1313.    -   c. Target fluorescent emissions cannot be inadvertently        triggered by the (excitation filtered) control LED 1311. This        error condition would otherwise lead to the control photosensor        1317 receiving unwanted contemporaneous signals from both the        target and control fluorophores.    -   d. Control fluorescent emissions cannot be inadvertently        triggered by the (excitation filtered) target LED 1331. This        error condition would otherwise lead to the target photosensor        1337 receiving unwanted contemporaneous signals from both the        target and control fluorophores.

FIG. 21A plots experimental results demonstrating enhancement offluorescent signal output by varying the height of the objective lensabove the mirror. Briefly, fluorescent beads (Thermo Fisher Scientific,p/n G0300, Pittsburgh Pa.) were inserted into a microfluidic detectionchamber and the detection chamber mounted under the objective lens 315of the detector essentially as shown in FIG. 3A. Using a digitalmicrometer, the height of the detector head above the microfluidiccartridge was then varied to construct the plot. In a second pairedexperiment, the mirrored surface was removed. The solid line (2100)shows the effect of varying the lens height in the presence of a mirroron the heating block; the dotted line (2101) shows the effect of varyinglens height in the absence of a back mirror. As can be seen, thepresence of the mirror seems to shift the optimal emission maximumbehind the mirror plane (i.e. a composite of the real and virtualfluorescent emissions captured in the lens).

FIG. 21B graphs the integrated output signal strength with (2103) andwithout (2102) the back mirror. Output signal was found to be optimizedby focusing the excitation beam at a focal point behind the mirror asshown in FIG. 15 and collecting emissions at a shorter working distanceas shown in FIG. 16.

In a second experiment, the detection chamber is filled with a liquidsample containing a soluble fluorophore and pumped through the chamberat constant rate so as to avoid quenching artifact. The detector headheight is then varied as before and the optimal detector heightdetermined. In related experiments, the working distance of the sensorlens and objective lens are also varied so as to optimize sensitivityand limit of detection. We learn that optimal configuration is notachieved when the objective lens is centered in the detection chamberand the other lenses are made confocal. When a mirror is used, decoupledoptics achieve advantageous results, a technological advance in thefield.

FIG. 22 shows scanning data collected for a molecular beacon hybridizedto an amplicon. The scanning axis transects detection wells (2200,2201)representing positive and negative test conditions respectively, and itcan be seen that signal is limited to the detection wells. In thefigure, the sample is scanned repetitively as the temperature in thedetection chamber is systematically varied. The scans are overlaid inthe plot to illustrate the spatial resolution of the data. Fluorescencescans for 35° C., 65° C., 70° C., 75° C. and 80° C. test conditions aremarked. Test plots at 40, 45, 50, 55, and 60° C., and the 85 and 90° C.plots were not well differentiated, as expected, and are notindividually marked. It can be seen that fluorescent signal is afunction of temperature. Fluorescence quenching is observed to increaseas the double stranded probe-target is melted, ie. signal is greatest at35° C. and is essentially not present at 80° C. In FIG. 23A, the data isplotted for signal versus temperature for the positive (2301, solidline) and negative (2302, dotted line) test conditions. In FIG. 23B, afirst derivative is plotted, indicating a FRET melt temperature of about70° C.

EXAMPLE I

In this example, the apparatus of the invention is shown to be useful indiagnosis of infectious disease by detection of the nucleic acids of apathogen in a human sample such as blood. Using on-board dry and liquidreagents, a blood sample is processed and DNA associated with Plasmodiumfalciparum is detected in about 30 minutes or less. DNA purified fromthe sample is subjected to PCR using two microfluidic chambers with dualtemperature zones as described in U.S. Pat. No. 7,544,506 and U.S.patent application Ser. No. 11/562,611 (Microfluidic Mixing andAnalytical Apparatus), which are coassigned. Amplicon is then detectedusing a FAM fluorescence-tagged molecular beacon directed at theamplified target. Optionally, a control consisting of a CaliforniaRed-tagged RNAaseP leukocyte exon sequence, with multiplexamplification, is used to validate the assay. A representative thermalmelt curve obtained using the thermo-optical interface of the inventionis shown in FIG. 22A.

EXAMPLE II

The apparatus of the invention is useful in the diagnosis ofcoagulopathies. Using on-board dry and liquid reagents, a blood sampleis assayed for Coagulation Factor VIIa deficiency by incubating plasmawith a fluorogenic substrate such as(D-Phe-Pro-Arg-ANSNH-cyclohexyl-2HCl; F.W.=777.81, HaematologicTechnologies, Essex Junction Vt.) where ANSN is fluorophore6-amino-1-naphthalene-sulfonamide, which lights up when the amide bondbetween the dye and the peptide is cleaved. Tissue Factor (TF) isobtained from Calbiochem (LaJolla Calif.) and incorporated intophosphatidylcholine or phosphatidylserine vesicles before use. TF isused in excess. A 100 uL substrate reaction mixture consisting of 20 mMHepes buffer, pH 7.4, 0.15 M NaCl, with 5 nM TF and containing 20 uMEDTA is incubated with a plasma sample for 10 min to form the activeenzyme complex. The ANSH substrate is then added. The rate of hydrolysisof substrate is linear over the normal range of Factor VIIa, and can bedetermined from a standard curve. Descriptions of assay development maybe found in US Patent Application 2009/0325203 and other experimentalliterature.

While the above is a description of the preferred embodiments of thepresent invention, it is possible to use various alternatives,modifications and equivalents. Therefore, the scope of the presentinvention should be determined not with reference to the abovedescription but should, instead, be determined with reference to theappended claims, along with their full scope of equivalents. Theappended claims are not to be interpreted as includingmeans-plus-function limitations, unless such a limitation is explicitlyrecited in a given claim using the phrase “means for.”

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

1. An apparatus for receiving a microfluidic cartridge and performing afluorescence assay on a liquid sample in one or more detection chambersof said microfluidic cartridge, the apparatus comprising: a) an inclinedmounting plate with pneumatic distribution manifold, said pneumaticdistribution manifold for distributing positive and negative pneumaticpressure to a pneumatic interface port adapted for sealingly interfacingwith said microfluidic cartridge, and having a thermal module attachedthereto, said thermal module having one or more heating elements withsurfaces configured for contactingly interfacing with said microfluidiccartridge, at least one said heating element having a surface modifiedas a mirror face for forming a thermo-optical interface with saidmicrofluidic cartridge; b) a floating stage mounted on said inclinedmounting plate, said floating stage with chassis and docking bay forreceiving said microfluidic cartridge therein and a clamping mechanismfor reversibly urging said microfluidic cartridge to contactingly engagewith said thermal module and pneumatic interface port; and c) a scanningdetector head with one or more optical channels and drive train, eachoptical channel with an objective lens, wherein said scanning detectorhead is slideably mounted on a pair of guiderails on said floating stageand powered for scanning across said one or more detection chambers of amicrofluidic cartridge in said docking bay, and is configured with anembedded microprocessor for detecting and processing fluorescent signalsemitted therein.
 2. The apparatus of claim 1, wherein said mirror faceis configured for contactingly opposing a thermo-optical window of saidmicrofluidic cartridge in said docking bay when urged to do so by saidclamping mechanism and said scanning detector head is configured forreflectively transilluminating said detection chamber when scanning onsaid pair of guiderails, said thermo-optical window comprising anoptically transparent compliant film with low resistance to thermaltransfer.
 3. The apparatus of claim 1, wherein said mirror face has anoptical flatness and reflectivity for increasing thermal conductivityand for increasing efficiency of fluorescent emission, excitation andcapture.
 4. The apparatus of claim 1, wherein said heating element withmirror face and scanning detector head with one or more optical channelsare configured for scanning said detection chamber of said microfluidiccartridge in said floating stage while adjusting or controlling thetemperature of said liquid sample.
 5. The apparatus of claim 4, whereinsaid one or more optical channels comprises a first optical channel anda second optical channel, said first optical channel comprising a firstsource LED, first source lens, first excitation filter, first dichroicbeamsplitter, first objective lens, first emission filter, first sensorlens, first sensor and first high gain amplifier tuned to the detectionof a first fluorophore in said liquid sample, and said second sensorchannel comprising a second source LED, second source lens, secondexcitation filter, second dichroic beamsplitter, second objective lens,second emission filter, second sensor lens, second sensor, and secondhigh gain amplifier tuned to the detection of a second fluorophore insaid liquid sample, and wherein said LEDs, lenses, beamsplitters,excitation filters and emission filters are configured so that theexcitation light of each channel is essentially monochromatic and thepassbands of the emission filters of each channel emission from saidfirst fluorophore and said second fluorophore have no crosstalk.
 6. Theapparatus of claim 1, wherein said microfluidic cartridge is disposable,and contains fluid and reagents sufficient for one assay, and whereinsaid assay is pneumatically controlled and driven by a programmable hostcontroller.
 7. The apparatus of claim 1, wherein said inclined mountingplate is mounted at a theta angle of between 10 to 45 degrees and a tiltdetector is used to disable the apparatus if the orientation is out oftolerance.
 8. The apparatus of claim 1, wherein said scanning detectorhead comprises an amplifier for amplifying a voltage from said sensor,thereby generating an amplified voltage, and said embeddedmicroprocessor is operatively coupled with clock and firmware forconverting said amplified voltage to a digital signal and outputtingsaid digital signal to a programmable host controller of said apparatus.9. The apparatus of claim 8, wherein said embedded microprocessor isconfigured for strobing a source LED at a strobe rate and with aselectable pulse width configured to effectively filter electrical orambient noise.
 10. The apparatus of claim 8, wherein said scanningdetector head is operatively driven on said guiderails in a series oflinear steps by a stepper motor while scanning.
 11. The apparatus ofclaim 10, wherein said embedded microprocessor, while scanning adetection chamber, sums all positive digital signals for any amplifiedvoltages greater than a threshold value and all neutral digital signalsfor any amplified voltage less than a threshold value, and outputs thesum to said programmable host controller, whereupon said programmablehost controller reports a qualitative assay result for detection of atarget analyte, such that said assay result is independent of bubbleinterference.
 12. The apparatus of claim 8, wherein said programmablehost controller is configured for digitally receiving said digitalsignal and displaying or electronically outputting a result of saidfluorescence assay.
 13. The apparatus of claim 12, wherein saidfluorescence assay is an assay for a nucleic acid target or a proteintarget in a biological sample.
 14. The apparatus of claim 7, whereinsaid inclined mounting plate is mounted at a theta angle of about 15degrees.